LiveCellMiner: A new tool to analyze mitotic progression

Live-cell imaging has become state of the art to accurately identify the nature of mitotic and cell cycle defects. Low- and high-throughput microscopy setups have yield huge data amounts of cells recorded in different experimental and pathological conditions. Tailored semi-automated and automated image analysis approaches allow the analysis of high-content screening data sets, saving time and avoiding bias. However, they were mostly designed for very specific experimental setups, which restricts their flexibility and usability. The general need for dedicated experiment-specific user-annotated training sets and experiment-specific user-defined segmentation parameters remains a major bottleneck for fully automating the analysis process. In this work we present LiveCellMiner, a highly flexible open-source software tool to automatically extract, analyze and visualize both aggregated and time-resolved image features with potential biological relevance. The software tool allows analysis across high-content data sets obtained in different platforms, in a quantitative and unbiased manner. As proof of principle application, we analyze here the dynamic chromatin and tubulin cytoskeleton features in human cells passing through mitosis highlighting the versatile and flexible potential of this tool set.


S1 Note: Experimental Details
LSM5live Experiments. LSD1 Data Set [1] HeLa cells expressing H2B-mCherry and α-tubulin-EGFP were transfected with the indicated siRNA oligonucleotides and seeded in eight-well µ-slide chambers (Ibidi). Starting at 30 h post-transfection, cells were imaged using a Plan-Apochromat 10× NA 0.45 objective and a 561-nm diode laser on a LSM5 live confocal microscope (Zeiss) equipped with a heating and CO2 incubation system (Ibidi). ZEN software (Zeiss) was used to acquire images from five 7.5-µm-spaced optical z-sections at various xy positions every 3 min. Single position *.ome files were generated from the maximum intensity projections in ZEN and converted into image sequences with Fiji software.

LSM5live Experiments. RecQL4 Data Set [2]
HeLa cells expressing H2B-mCherry and EGFP-α-tubulin were transfected with the indicated siRNA oligonucleotides in eight-well µ-slide chambers (Ibidi) and, after 24 h, were imaged for 48 h in a LSM 5 live confocal microscope (Zeiss) equipped with a heating and CO2 incubation system (Ibidi). Seven 3.6-µm-spaced optical z-sections at various positions every 3 min were acquired with a Plan-Apochromat 20× NA 0.8 objective and a 488-nm and 561-nm diode lasers controlled by ZEN software. For the analysis, maximum intensity projections in Z were generated in ZEN for every position and converted into temporal image sequences with the free licensed AxioVision software (LE64; V4.9.1.0).

LSM710 Experiments. CTRL vs PP2A Data Set (Unpublished)
HeLa cells expressing H2B-mCherry were transfected with the indicated siRNA oligonucleotides in eight-well µ-slide chambers (Ibidi) and, after 24 h, were imaged for 48 h in a LSM 710 confocal microscope (Zeiss) equipped with an Incubator XL S1(Zeiss). Seven 2.6-µm-spaced optical z-sections at various positions every 3 min were acquired with a Plan-Apochromat 20× NA 0.8 objective and a 488-nm Argon and 561-nm DPSS lasers controlled by ZEN software (Zeiss). For the analysis, maximum intensity projections in Z were generated for every position and converted into temporal image sequences in ZEN 2.3 software (Zeiss). Nikon Experiments. VPS72, INO80, SRCAP, EP400 and H2A.Z. Data Set [3] HeLa cells expressing H2B-mCherry were transfected with the indicated siRNA oligonucleotides in eight-well µ-slide chambers (Ibidi) and, after 48 h, were imaged for 48 h with the widefield module of a Ti2 Eclipse (Nikon) equipped with a LED light engine SpectraX (Lumecor) and GFP/mCherry filter sets, a Plan-Apochromat 10x NA 0.5 objective and environmental control system (Ibidi). Elements software (Nikon) was used to perform fluorescence multi-position imaging every three minutes and the subsequent conversion to image sequences.

Nikon Experiments. RPE cells. Data Set [3]
RPE cells expressing H2B-mCherry were transfected with 20nM of the indicated siRNA oligonucleotides in eight-well µ-slide chambers (Ibidi) and, after 48 h, were imaged for 48 h with the widefield module of a Ti2 Eclipse (Nikon) equipped with a LED light engine SpectraX (Lumecor) and GFP/mCherry filter sets, a Plan-Apochromat 20x NA 0.75 air objective and environmental control system (Ibidi). Elements software (Nikon) was used to perform fluorescence multi-position imaging every three minutes and the subsequent conversion to image sequences.

Screening Data Set by Hériché et al. [4]
The screening data set is publicly available at Image Data Resource (IDR) (https://idr.openmicroscopy.org/webclient/?show=screen-102). HeLa cells stably expressing HIST1H2BJ-mCherry and LMNA-eGFP in each well of siRNA-coated 96-well plates. The images were acquired with an Olimpus IX-81 automated epifluorescence microscope with a 20× objective and a time interval of 8.5 min for 44 h. Four independent replicates were acquired for each siRNA treatment.